lncRNA在半滑舌鳎孕激素通路中的调控功能及其对卵巢成熟过程的作用

Progestin regulation of oocyte maturation is a vital event in the reproduction of fish. The interaction between the mode of progestin regulation and the environment determines the maturation levels of fish ovary. Progestin hormone induces oocyte maturation through the membrane progestin receptors (mPRs). The relative abundance of mPRs on the oocyte membrane is clossly associated with the process of oocyte maturation. Recent studies showed that the long non-coding RNA (lncRNA) plays very important role in regulating various physiological processes. Furthmore, lncRNA regulates essential pathways that contribute to ovarian development and maturation in human and mouse. However, there is no information about function and regulation mechanism of lncRNA of the progestin pathway during oocyte maturation of fish. In the past several years, we have been supported by NSFC funding to study the physiological function of mPRs in Cynoglossus semilaevis. On the basis of our previous studies of mPRs, this project will further explore the lncRNA function in the ovary maturation. The dynamic expression profiles of lncRNA along the brain-pituitary-ovary axis will be constructed by RNA-seq analysis during the ovary maturation of Cynoglossus semilaevis. In the progestin pathway, the crucial lncRNAs will be selected. And, their response mechanism to the exogenous gonadotropin stimulation will be uncovered by real time PCR and in situ hybridization. The molecular regulatory mechanism of crucial lncRNA will be explored in the pathway of progestin shift and mPRs transportation to the cell membrane. These relevant findings may illuminate the regulation function of lncRNA in the progestin pathway and its role during ovary maturation of Cynoglossus semilaevis. In addition, the results of this project will hopefully lay a theoretical foundation for exploring the molecular mechanism of low levels of maturity of ovary in the cultured fish. And, it will provide scientific basis for guiding the regulation of broodstock reproduction.

孕激素调控卵母细胞成熟是鱼类繁殖的一个关键环节，其调控模式与环境的互作共同决定了鱼类卵巢成熟度。孕激素通过膜孕激素受体（mPRs）诱导鱼类卵母细胞成熟，其表达丰度与卵母细胞发育成熟进程密切相关。长链非编码RNA（lncRNA）在动物卵巢发育成熟过程中具有重要作用，但在鱼类卵巢成熟过程中孕激素通路的作用机制尚未明确。本项目在前期对半滑舌鳎mPRs生理功能研究的基础上，拟采用RNA-seq绘制半滑舌鳎卵巢成熟过程中脑-垂体-卵巢轴的lncRNA动态表达谱；筛选孕激素通路上的关键lncRNA，揭示其在卵巢和血清中对外源激素刺激的响应机制；探索这些关键lncRNA在孕激素通路转换和mPRs转运过程中的调控机理；为阐明lncRNA在半滑舌鳎孕激素通路中的调控功能及对卵巢成熟过程的作用奠定基础。此外，本项目的实施可望为探究养殖鱼类卵巢成熟度低的的分子机制奠定理论基础，并为指导亲鱼生殖调控提供科学依据。

lncRNAs参与多种生物学过程。然而，lncRNAs在半滑舌鳎繁殖过程中的作用机制尚不清楚，特别是在调控卵巢发育成熟和排卵方面。对3个不同卵巢发育期的脑-垂体-性腺-肝脏轴组织进行了转录组测序分析。在脑和垂体分别鉴定到5135和5630个DE mRNAs，分别鉴定到378和532个DE lncRNAs。通过富集分析解析了 DE mRNAs和lncRNAs的功能，揭示了它们参与新陈代谢、信号转导和内分泌信号。基于反义、顺式和反式调控机制构建了LncRNA-靶基因相互作用网络。构建了竞争性内源 RNA 网络。在卵巢组织的发育IV期与V期比较组， 鉴定到312个DE mRNAs 和58个DE lncRNAs。 在卵巢V期与VI期比较组， 鉴定到1059个DE mRNAs和187个DE lncRNAs。 GO和KEGG富集分析表明DE mRNAs富集在ECM受体相互作用、卵母细胞减数分裂和类固醇激素生物合成通路。GSEA分析进一步确定了潜在的繁殖相关通路。预测到有几种lncRNAs可调节繁殖相关基因。功能性的富集分析lncRNAs的这些靶基因揭示了它们参与在多种信号通路和繁殖相关通路如孕酮介导的卵母细胞成熟和卵母细胞减数分裂。XR_522278.2 和 XR_522171.2主要表达在卵巢。XR_522278.2 和 XR_522171.2分别与其靶基因 cyp17a1和 cyp19a1共定位在滤泡细胞层。激素调控后，卵母细胞发育成熟相关的基因及其对应的lncRNAs 表达水平升高。共鉴定到21,599个circRNAs。一些DE circRNAs的亲本基因与生物学过程中的关键信号通路密切相关，可能在卵巢发育和成熟过程中发挥重要作用。筛选到的circRNAs 参与了10 个信号通路。 验证了circRNAs为环型结构，且能抵制RNase R消化的作用。共鉴定到 3224 个miRNAs。鉴定出的关键miRNAs在不同卵巢发育期同时调控几种mRNAs 并参与到不不同的生物学功能。通过KEGG通路分析，发现了8个富集的重要信号通路。验证了igf2r存在miR-186-x结合的位点。这些发现表明lncRNAs 、circRNAs、miRNAs广泛存在于半滑舌鳎繁殖相关的组织，并在卵巢发育和卵母细胞成熟过程中具有潜在的重要调控作用，为提高半滑舌鳎的繁殖性能提供了理论依据。